Multi-guild microbial cooperation sustains long-term anaerobic toluene degradation through sulfur cycling

Anaerobic degradation of aromatic hydrocarbons such as toluene plays a critical role in the natural and engineered attenuation of contaminated environments. Here, we developed and characterized a microbial consortium enriched under strictly anoxic conditions, capable of sustained toluene degradation through sulfate reduction. By integrating biodegradation kinetics, long-read 16S rRNA profiling, and genome-resolved metagenomics, we elucidated the structure and function of a multi-guild community. The consortium was co-dominated by <i>Desulfoprunum</i>, a sulfate-reducing bacterium (SRB), and <i>Sulfurovum</i>-affiliated sulfur oxidizers (~34% each), with additional members including <i>Stenotrophomonas, Achromobacter</i>, and <i>Stutzerimonas</i>. Such co-dominance appears uncommon, as sulfate-reducing enrichments are often characterized by low diversity and the predominance of a single lineage, such as <i>Desulfobacula</i> or <i>Desulfosarcina</i> in marine systems. Genome-resolved analyses recovered seven metagenome-assembled genomes (MAGs) with distinct but complementary metabolic roles. <i>Desulfoprunum</i> encoded the fumarate-addition pathway (<i>bss/bbs</i>) for anaerobic toluene activation and dissimilatory sulfate reduction (<i>aprAB, dsrAB</i>). In contrast, <i>Sulfurovum</i> and several <i>Gammaproteobacteria</i> encoded sulfide:quinone oxidoreductase (<i>sqr</i>), coupling H₂S detoxification to energy conservation, while a <i>Moranbacterales</i> MAG carried a putative sulfhydrogenase (<i>hydAB</i>) potentially catalyzing elemental sulfur (S°) reduction. Additional MAGs encoded assimilatory sulfate reduction (<i>cys</i>), suggesting integration of sulfur into biosynthetic pathways. Together, these features are consistent with the presence of a putative distributed sulfur redox loop, in which biogenic H₂S may be recycled via oxidation and reduction reactions mediated by co-occurring taxa. This sulfur loop is hypothesized to contribute to buffering sulfide toxicity and stabilize redox dynamics, thereby potentially supporting long-term toluene degradation under sulfidic conditions. Our findings highlight anaerobic degradation as a community-driven process enabled by sulfur-cycling interactions. By revealing the role of cryptic sulfur cycling in stabilizing hydrocarbon degradation, this work offers a new framework for designing bioremediation strategies in contaminated anoxic environments.